Glucagon-like peptide-1 (GLP-1; 30 amino acids) is secreted from enteroendocrine cells of the distal small intestine in response to food ingestion (1). GLP-1 plays an important role in glucose metabolism and homeostasis by inhibiting gastric emptying, glucagon secretion, and glucose production (2). In addition, GLP-1 induces insulin release from the pancreatic β-cells as well as their proliferation. The GLP-1 receptor (GLP-1R) has been identified in normal tissues such as the pancreatic β-cells, stomach, brain, and lung, and it has been shown to be highly overexpressed in human insulinomas and gastrinomas (3). In insulinomas, GLP-1R density is considerably greater, and the GLP-1R is more frequently observed than somatostatin receptors.
Exendin-4 is a GLP-1 analog with 39 amino acids isolated from the venom of the Gila monster (Heloderma suspectum) (4). Exendin-4 and GLP-1 share a 53% amino acid sequence homology. Exendin-4 is a more potent and longer-lasting GLP-1R agonist than GLP-1. Exendin-4 is resistant to cleavage by dipeptidyl peptidase IV, whereas the first two N-terminal amino acids of GLP-1 are rapidly cleaved. Exenatide, a synthetic version of exendin-4, is the first GLP-1 mimetic approved by the United States Food and Drug Administration (FDA) for use in select patients with type 2 diabetes (5). 111In-Labeled diethylenetriamine pentaacetic acid-aminohexanoic acid-Lys40-exendin-4 (111In-DTPA-Ahx-Lys40-exendin-4) has been developed for single-photon emission computed tomography (SPECT) imaging of the GLP-1R (6).
For optical near-infrared (NIR) fluorescence imaging, Reiner et al. (7) replaced the Lys at position 12 of exendin-4 with the non-natural amino acid (S)-2-amino-4-pentynoic acid to form a neopeptide, E4X12. The azide-functionalized NIR dye VivoTag-S 750 (VT750) was conjugated to the non-natural amino acid at position 12 via azide-alkyne cycoaddition to form VT750-(S)-2-amino-4-pentynoic acid12-exendin-4 (E4X12-VT750). E4X12-VT750 has been evaluated as an optical imaging agent for GLP-1R density and β-cell mass (BCM) in normal mice and in mice treated with streptozotocin (STZ) to induce BCM loss and diabetes.
11-Azido-3,6,9-trioxaundecan-1-amide-VT750 (azido-VT750) was prepared by incubation of 11-azido-3,6,9-trioxaundecan-1-amine (46 µmol) and VT750 (8.6 µmol) for 3 h at room temperature (7). Azido-VT750 was purified with high-performance liquid chromatography (HPLC), with a chemical yield of 36%. Azido-VT750 (1.2 µmol) was added to an aqueous solution (pH 7.2) of E4X12 (0.8 µmol), ascorbic acid (2.5 mmol), and CuSO4 (2.5 mmol). The mixture was incubated for 18–20 h at room temperature. E4X12-VT750 was isolated with HPLC, with a chemical yield of 64%. E4X12-VT750 was identified with HPLC and matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). There was one VT750 dye per conjugate. E4X12-VT750 exhibited an absorption maximum at 754 nm and an emission maximum of 774 nm.
In Vitro Studies: Testing in Cells and Tissues
Cellular accumulation of E4X12-VT750 (0, 0.01, 0.1, 1, 10, and 100 nM) was measured in mouse insulinoma MIN6 (GLP-1R–positive), HEK/hGLP-1R (human GLP-1R–positive), and NIH3T3 fibroblast (GLP-1R–negative) cell lines (7). There was a dose-dependent increase in cellular fluorescence signals in MIN6 and HEK/hGLP-1R cells with a 50% effective dose of 3.3 nM in both cell lines for 90 min at 37°C. However, no signal was observed in NIH3T3 cells, even at 100 nM of E4X12-VT750. Preincubation with various concentrations of exendin-4 for 60 min before the addition of 10 nM E4X12-VT750 decreased the fluorescence signals in MIN6 and HEK/hGLP-1R cells with a 50% inhibition concentration of ~3 nM. Fluorescence microscopy showed that E4X12-VT750 was internalized into the vesicular compartments of HEK/hGLP-1R cells. The cell-surface binding and internalization of E4X12-VT750 were inhibited with excess exendin-4.
Reiner et al. (7) performed intravital confocal imaging of pancreatic islets and β-cells in mice with the mouse insulin 1 promoter green fluorescence protein (MIP-GFP). NIR fluorescence signal was observed in β-cells within 10 min after intravenous injection of 2–4 nmol E4X12-VT750, and the signal gradually increased to ~64-fold more than background at 120 min. Injection of exendin-4 (15 nmol) 20 min before injection of E4X12-VT750 reduced the signal to background level in β-cells at 60 min after injection. E4X12-VT750 exhibited a weighted blood half-life of 84 s. E4X12-VT750 fluorescence signal colocalized with GFP signal in β-cells (R2 = 0.944). NIR fluorescence signal was also observed with microendoscopic imaging of the pancreata of anesthetized mice. Ex vivo NIR fluorescence distribution in organs of normal mice at 60 min after E4X12-VT750 injection was compared with the autofluorescence of noninjected mice. The organ with the highest fluorescence was the kidney (69%), followed by the liver (15%), pancreas (8%), intestine (4%), lung (3%), stomach (2%), spleen (<1%), and heart (<1%). E4X12-VT750 was mostly eliminated by the kidneys and liver.
Ex vivo studies of pancreatic accumulation of E4X12-VT750 were performed in normal mice (n = 4–5/group) treated with various doses (0, 75, 150, and 500 mg/kg) of STZ to induce BCM loss and diabetes. Pancreatic fluorescence signal was obtained as fluorescence/volume, and BCM was determined with histo-immunological staining of pancreas sections at 60 min after injection. Mice exhibited normal glucose levels with 75 mg/kg STZ and became hyperglycemic with 150 and 500 mg/kg STZ. There was a dose-dependent loss of NIR fluorescence signals in the pancreata from the STZ-treated mice. There was a good correlation (R2 = 0.854, P < 0.0001) between loss of fluorescence signal and BCM.
Other Non-Primate Mammals
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Created: November 1, 2011; Last Update: January 5, 2012.
National Center for Biotechnology Information (US), Bethesda (MD)
Leung K. VivoTag-S 750-(S)-2-amino-4-pentynoic acid12-exendin-4. 2011 Nov 1 [Updated 2012 Jan 5]. In: Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.